Project Summary/Abstract The lung is a portal of entry for a variety of pathogenic organisms that may cause pneumonia or even the acute respiratory distress syndrome (ARDS), which is a life-threatening syndrome that has no specific treatments except for the provision of gentle mechanical ventilation and supportive care. The neutrophil is a critical cellular mediator of the early immune response to pathogenic lung infections, but an overly exuberant neutrophil response may cause collateral damage to the lung and lead to acute lung injury. In this application, a new function of neutrophils, the release of neutrophil extracellular traps (NETs), will be investigated in mouse models of acute lung infection. NETs are composed of extracellular chromatin decorated with neutrophil granular proteins and have been proposed to serve an important role in the trapping and killing of bacteria and other pathogens. However, we propose that NETs are toxic to adjacent cells and are on balance a maladaptive and dispensible host response in pneumonia and acute lung injury. Aim 1 of this application will investigate the role of NETs in mouse models of bacterial and viral pneumonia using novel techniques to visualize and quantify NETs. We will also test the role of lipoxin mediators signaling through Fpr2 on neutrophils in regulating NET production and the progression to acute lung injury. Aim 2 will test the role of DNase1 in regulating the degradation of NETs in the lung and the important effects of DNase1 treatment on the containment of infection and preservation of the lung barrier. In translational studies, Aim 2 will also use biological samples from critically ill patients with severe infections to test if NETs and DNase1 bioactivity (a) predict the progression to ARDS or (b) are associated with poor clinical outcomes in ARDS. The factors that determine whether a localized lung infection progresses to acute lung injury are not known, but this application proposes that NETs are critical mediators in this process and targetable. Our studies using live bacterial and viral infections, novel approaches to visualize acute lung inflammation and injury, and NETs, pharmacologic and genetic approaches to neutralize NETs, and translational studies to complement our mouse models, are well-positioned to provide definitive evidence on the in vivo significance of NETs, and to position NETs as a novel target for the treatment of pathogen-induced lung injury.